Engineered proteins combining several functional protein domains have been proposed as therapeutic agents to address major medical challenges, in particular, for selective killing of diseased cells, such as cancer cells and cells infected by bacteria or viruses, and for generation of protective immune responses. One such type of multi-domain protein, referred as a “sitoxin” (for “signal-regulated cleavage-mediated toxin”), comprises a toxin effector domain, a domain including an intracellular signaling moiety, and an intervening domain positioned between the aforementioned domains, which includes a cleavage site for a predetermined protease expressed within a target cell (Varshaysky, 1995).
A diphtheria toxin-based sitoxin containing a signal for N-end-rule-mediated degradation upstream of a cleavage site for the HIV type 1 protease has been disclosed, and reportedly exhibited a substantial increase in toxicity via inhibition of cellular protein synthesis following in vitro cleavage by the viral protease, although such toxins were unable to selectively eradicate HIV-1-infected cells (Falnes et al., 1999).
The term “zymogenization” has been used to describe modification of an enzyme so as to convert a constitutively active enzyme to a protease-activatable form. Modified forms of bovine RNase A have been disclosed, in which the enzyme was modified so as to expose its natively conformed active site only upon proteolytic cleavage mediated by proteases of Plasmodium falciparum, HIV or HCV, which elicited significant enhancement of in vitro RNase activity (Plainkum et al., 2003; Johnson et al., 2006; Turcotte and Raines, 2008).
An engineered form of Vip2, an actin modifying vegetative insecticidal protein produced by the bacterium Bacillus cereus, has been disclosed in which a propeptide was fused to the C-terminus of Vip2, so as to form a Vip2 proenzyme with significantly reduced enzymatic activity, while having the ability to express as a transgene in corn plants. According to the disclosure, the proenzyme was activated extracellularly within the digestive tract of the pest western corn rootworm, and resulted in the killing of rootworm larvae (Jucovic et al., 2008).
Pseudomonas aeruginosa exotoxin A (PE; also referred to as Pseudomonas exotoxin or ETA) is a three-domain bacterial toxin that kills mammalian cells by gaining entry to the cytosol and inactivating protein synthesis. PE is composed of three major domains and one minor domain. Domain 1a (amino acid residues 1-252) is the cell-binding domain; domain 2 (amino acid residues 253-364) is the translocation domain that enables PE to reach the cytosol, and domain 3 (amino acid residues 395-613) has ADP-ribosyl transferase activity that inactivates translation elongation factor 2 and thus causes cell death. The pathway of toxin entry to mammalian cells includes the steps: 1) binding to a surface receptor, mediated by the binding of PE domain 1 to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP) which is ubiquitously expressed in most tissues and cell types (Gu et al., 1996); 2) internalization via coated pits and endosomes, and 3) proteolytic cleavage between Arg-279 and Gly-280 within domain 2 and reduction of disulfide bonds, which is mediated by the cellular protease furin, and generates the active C-terminal fragment (residues 280-613) (FitzGerald and Pastan, 1991). At the final step 4), the enzymatically active C-terminal fragment is translocated by retrograde transport through the trans-Golgi network, then backward through the Golgi apparatus to the endoplasmic reticulum and from there to the cytosol. Once in the cytosol, this fragment inhibits protein synthesis by ADP ribosylation of elongation factor 2 (Iglewski and Kabat, 1975).
Diphtheria toxin (DT), produced by Corynebacterium diphtheriae, kills mammalian cells via a mechanism similar to that of PE, namely, by gaining entry to the cytosol and inactivating protein synthesis by ADP ribosylating elongation factor 2. X-ray crystallographic analysis demonstrated that DT is composed of three structural domains: the amino terminal catalytic (C) domain (also referred as “DTA”) the translocation domain (T) and the carboxy-terminal receptor binding (R) domain. DT is cleaved at the surface of sensitive eukaryotic cells by the enzyme furin, and following binding to its receptor (the heparin binding epidermal growth factor-like precursor), the di-chain protein that is linked by a single disulfide bond is internalized into clathrin coated pits and reaches the lumen of a developing endosome. Upon acidification, the T domain facilitates the translocation of the catalytic domain directly across the endosomal membrane and into the host cell cytoplasm (Rafts and Murphy, 2004; Deng and Barbieri, 2008).
A large group of toxins naturally found in plants are classified as ribosome-inactivating proteins (RIPs), which are polynucleotide adenosine glycosidases that cleave the glycosidic bond of an adenosine base in an evolutionarily conserved sequence (GAGA) in the alpha-sarcin/ricin loop (SRL) of 28S rRNA of eukaryotic ribosomes. This depurination reaction prevents binding of elongation factor 2 to the eukaryotic ribosome, and results in protein synthesis inhibition. RIPS include ricin, abrin, Shiga-like toxin 1 (SLT-1), modecin, volkensin, visumin, trichosanthin, maize RIP, luffaculin 1, and alpha-luffin (for a review see for example, Peumans et al, 2001). RIPs are sub-classified according to their molecular structure as: Type I, consisting of a single polypeptide chain corresponding to an N-glycosidase domain; Type II, consisting of two polypeptide chains in disulphide linkage, corresponding to an N-terminal N-glycosidase domain and a C-terminal lectin domain, which are synthesized from a single precursor that undergoes post-translational processing; or Type III (or alternately termed two-chain Type I), consisting of two polypeptide chains, corresponding to segments of an N-glycosidase domain, held together by non-covalent interactions which are synthesized from a single precursor that undergoes post-translational processing.
Ricin toxin (RT) from Ricinus communis is a type II RIP consisting of the catalytic A chain covalently linked to the lectin domain B chain, the latter of which binds to galactose residues on the surface of eukaryotic cells and stimulates receptor-mediated endocytosis of the toxin molecule (Stirpe and Battelli, 2006). Cell-bound ricin is taken up by endocytosis, following which most of the toxin molecules are recycled back to the cell surface or transported to the lysosomes and degraded. A small fraction is translocated by retrograde transport to the trans-Golgi network, backward through the Golgi apparatus to the endoplasmic reticulum and from there to the cytosol (Olsnes, 2004).
Maize RIP is a Type III RIP produced as an inactive precursor proenzyme (pro-RIP) having a 25-amino acid internal inactivation region on the protein surface. During germination, proteolytic removal of this internal inactivation region generates the active heterodimeric maize RIP (Walsh et al., 1991; Bass et al., 1992). HIV-activated toxins designed on the basis of maize RIP have been disclosed (Law et al., 2010). According to this disclosure, replacement of the first and last 10 residues of the internal inactivation region with two HIV-PR recognition sequences, and fusion of an 11 amino acid transduction peptide derived from the HIV-1 Tat protein to the N-termini of the modified RIPs resulted in the generation of pro-RIPs which were efficiently cleaved in-vitro by recombinant HIV-PR or in-vivo (in HIV infected cells) by virally encoded protease. Upon treatment of infected cells, the N-glycosidase and anti-viral activities of the modified cleavable RIPs were found to be higher than these of an uncleavable-nonactivated pro-RIP and resembled these of an activated mutant in which the inhibitory region was genetically removed.
The mammalian defensins are a family of small cationic peptides characterized by their β-sheet-dominant structure stabilized by two or three intramolecular disulfide bonds (Ganz, (2003) Nat. Rev. Immunol. 3, 710-720). They are further subdivided into three subfamilies: α-defensins; β-defensins; and θ-defensins. Six human α-defensin peptides (each composed of 29-35 amino acid residues) have been identified from five genes, namely HNP (human neutrophil peptide)-1 to -4, HD-5, and HD-6 (Ericksen et al., Antimicrob Agents Chemother. 2005; 49:269-75). HNP-1-4 are expressed primarily by granulocytes and certain lymphocyte populations (Ganz et al., (1985) J. Clin. Invest. 76, 1427-1435; Agerberth et al., (2000) Blood 96, 3086-3093). The amino acid sequences of HNP-1-3 are identical except for the first N-terminal residue. The α-defensins have been identified as natural peptide antibiotics which display microbicidal activity against numerous bacteria, fungi, and viruses (Lehrer et al., (1993) Annu. Rev. Immunol. 11, 105-128).
HNP-1, HNP-2 and HNP-3 have been disclosed to have neutralizing activity against lethal factor (LF), the major toxin of B. anthracis (Kim et al., Proc Natl Acad Sci USA. 2005 Mar. 29; 102(13)). HNP-1, HNP-2 and HNP-3 have further been disclosed to neutralize toxins of the mono-ADP-ribosyltransferase family, particularly diphtheria toxin and Pseudomonas exotoxin A (Kim et al Biochem J (2006) 399, 225-229). HNP-1, HNP-3, and enteric human defensin-5 (HD-5) have been disclosed to inhibit the activity of Clostridium difficile toxin B.
The conserved C-terminus of the ribosome stalk proteins P1 and P2 has been disclosed to interact with both ricin and Shiga-like toxin 1. This interaction is reportedly required for efficient ribosome binding and cytotoxicity. In addition, a synthetic peptide corresponding to the sequence of the conserved C terminus of P1 and P2 was shown to inhibit the ribosome-inactivating function of SLT-1 (Vater et al., 1995; Chiou et al., 2008; McCluskey et al., 2008). The crystal structure of a similar peptide in complex with the type I RTP trichosanthin has been disclosed (Too et al., 2009).
Hepatitis C virus (HCV) is a small, enveloped RNA virus belonging to the Hepacivirus genus of the Flaviviridae family, which is recognized as a major cause of chronic liver disease and affects approximately 200 million people worldwide. Persistent infection is associated with the development of chronic hepatitis, hepatic steatosis, cirrhosis, and hepatocellular carcinoma. A protective vaccine for HCV is not yet available, and the currently favored treatment, which is a combination of pegylated α-interferon and ribavirin, fails to eliminate infection in nearly 50% of infected subjects.
The HCV genome encodes one large open reading frame that is translated as a polyprotein and proteolytically processed to yield the viral structural and non-structural (NS) proteins. The envelope glycoproteins E1 and E2 and the core protein are the structural proteins, which together form the viral particle. The non-structural proteins include the p7 ion channel, the NS2-3 protease, the NS3 serine protease/RNA helicase and its co-factor NS4A, the NS4B and NS5A proteins and the NS5B RNA-dependent RNA polymerase (RdRp) (Moradpour et al., 2007; Suzuki et al., 2007). HCV polyprotein processing involves the NS2-3 autoprotease, which cleaves in cis at the NS2-3 junction, and the NS3-4A serine protease, which cleaves at four downstream NS protein junctions, respectively termed NS3/4A; NS4A/4B; NS/4B/5A, AND NS5A/5B. NS3 has been extensively studied and shown to possess multiple enzymatic activities that are essential for HCV replication. The N-terminus, in complex with its co-factor NS4A, primarily functions as a serine protease, which cleaves the viral polyprotein precursor downstream to NS3. The remaining ⅔ of the protein has helicase and NTPase activities (Bartenschlager, 1999).
WO 2006/109196 discloses a method to identify a compound that inhibits HCV replication comprising: contacting a genetically modified mouse with a compound and analyzing the expression of NS3 protease activity, whereby a compound that inhibits expression of NS3 is indicative that said compound inhibits HCV replication.
WO 2009/022236 discloses compositions that comprise an isolated nucleic acid encoding a chimeric hepatitis C virus NS3/4A polypeptide or a fragment thereof, which comprises a sequence that encodes an antigen, preferably a non-HCV epitope, wherein the nucleic acid encoding the antigen can be inserted within the NS3/4A nucleic acid or attached thereto. Further disclosed is a composition comprising a recombinant peptide immunogen comprising an antigen, such as an antigen comprising an epitope from a plant, virus, bacteria, or a cancer cell; and a heterologous HCV NS3 protease cleavage site. According to the disclosure, NS3/4A is used as a carrier or adjuvant to provide T helper cells access to a fused antigen, thereby enhancing the immune response to the fused antigen, and the antigen may be inter alia a toxin.
WO 2008/052490 discloses a chimeric peptide containing at least one segment which inhibits the activation of the NS3 protease of a Flaviviridae family virus, and a cell penetrating segment which can inhibit or attenuate infection by the virus. Further disclosed are pharmaceutical compounds containing the chimeric peptides and use thereof for prevention and/or treatment of Flaviviridae virus infections.
WO 2004/005473 discloses an immunogenic fusion protein comprising (a) a modified NS3 polypeptide comprising at least one amino acid substitution to the HCV NS3 region, such that protease activity is inhibited, and (b) at least one polypeptide derived from a region of the HCV polyprotein other than the NS3 region. According to the disclosure, compositions of the invention may include a carrier or an adjuvant inter alia a detoxified mutant of a bacterial ADP-ribosylating toxin such as a cholera toxin.
WO 2003/064453 discloses active inhibitors, termed “trojan inhibitors” (TI) and the use thereof in the form of specifically shaped trojan proteasome-inhibitors (TPI) or trojan assembling-inhibitors (TAI), such as proteasome- and assembling-inhibitors which are initially inactive and are only activated in the target cell by means of a specific protease for the target cell. According to the disclosure, said inhibitor can be used in the treatment of viral infections, whereby a virus-specific protease is expressed, particularly in HIV-infections and AIDS-therapy, and in the therapy of tumoral diseases, whereby the tumor cells are characterized by a specific protease.
WO 2002/087500 discloses a synthetic prototoxophore, which is a relatively non-toxic compound that includes a toxin moiety, such as an antimetabolite or a DNA intercalating agent, and a substrate domain for a viral enzyme, which upon binding of a viral enzyme to the substrate domain, the catalytic activity of the viral enzyme converts the prototoxophore to a toxophore, which is toxic to a cell. Further disclosed are methods of using a prototoxophore to reduce or inhibit viral infectivity, and to ameliorate the severity of a viral infection.
WO 2005/090393 discloses a composition comprising a first effector component of a multimeric bacterial protein toxin, the first effector component comprising at least a first monomer and a second monomer, wherein said first and second monomers form a heterooligomer, wherein said first and second monomers are different, and each of said first and second monomers are modified by at least two of the following methods: (a) substitution of a native cell-recognition domain for a non-native cell-recognition domain; (b) substitution of a native proteolytic activation site for a non-native proteolytic activation site; (c) modification of said first monomer to generate a first modified monomer, whereby said first modified monomer can pair only with said second monomer; (d) modification of said first monomer and said second monomer, whereby a second effector component can bind only at a site formed by the interaction of said first monomer and said second monomer molecule; or (e) a combination thereof.
According to the disclosure, the bacterial protein toxin may be inter alia cholera toxin or Shiga toxin; (b) may comprise substitution of a native furin cleavage site by a serine protease cleavage site; and the composition may be used for treating a viral infection inter alia HCV.
WO 2000/062067 discloses a fusion molecule comprising at least one protein transduction domain (PTD) and at least one linked molecule, inter alia an anti-infective drug.
WO 1999/029721 discloses an anti-pathogen system comprising a fusion protein comprising a covalently linked protein transduction domain and a cytotoxic domain (i.e. a caspase), wherein the cytotoxic domain further comprises at least one pathogen-specific protease cleavage site, wherein the pathogen may be inter alia HCV.
U.S. Pat. No. 7,247,715 discloses a purified and isolated nucleic acid sequence having a nucleotide sequence encoding an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence linking the A and B chains, the heterologous linker sequence containing a cleavage recognition site for a protease, inter alia a viral protease, wherein the cleavage recognition site is recognized by the viral protease inter alia hepatitis C virus.
There remains an unmet need for therapeutic agents which are effective for the treatment of debilitating diseases, including viral infections such as HCV.